For many biological, biochemical, diagnostic or therapeutic purposes, it is necessary to accurately and precisely determine the amount or concentration of a nucleic acid in a sample. dPCR is a rather new approach to nucleic acid detection and quantification that offers an alternative method to conventional real-time quantitative PCR for absolute quantification of nucleic acids and rare allele detection. dPCR works by partitioning a sample of nucleic acids into many individual, parallel PCR reactions; some of these reactions contain the target molecule (positive) while others do not (negative). Following PCR analysis, the fraction of negative reactions is used to generate an absolute count of the number of target molecules in the sample. One of the key advantages of dPCR over real-time PCR is its superior accuracy of quantification. This advantage relies on inherent properties of dPCR as quantification only requires correct counting of positive partitions and the knowledge of the theoretical partition volume (the count number is not very sensitive to PCR efficiency). A quantification standard is not required. This eliminates potential quantification errors caused by the standard itself.
The prior art provides methods in order to identify incorrect positive or negative counts and for calibrating or normalizing signals in droplet-based assay (US 2013/0302792 A1). This normalization should improve the separation between positive and negative counts. Hence the normalization reduces the risk of false positive or negative counts. The ultimate goal is to improve the accuracy and precision of the determination of the nucleic acid concentration by correcting the signal obtained for the nucleic acid.
However, the methods of the prior art do not account for quantification errors in PCR due to situations, in which the true volume in the dPCR reaction areas differs from the expected or intended one.
Accordingly, there is a need for methods of quantifying a nucleic acid of interest by dPCR, which reduce quantification errors caused by reaction volume deviations. The object of the present disclosure was to provide those methods.